Production And Characterization Of Overexpression Of Melatonin Synthase AANAT/HIOMT Gene In Ovine

The melatonin(MT)participates in regulation of the immune and reproductive activities of animals through receptor signaling pathways as a neuroendocrine hormone and potent antioxidant.In this study,a transformed the melatonin synthetase sheep mammary gland bioreactor which can generate melatonin-enriching milk had been successfully developed.The mechanism of local melatonin signaling involved in inflammatory response,ovulation and embryo attachment was explored through the model.The transgenic ovine group carrying overexpressed melatonin synthetase gene arylalkylamine N-acetyltransferase(AANAT)and hydroxyindole o-methyl transferase(HIOMT)had been successfully established in this study.It was proved that the AANAT and HIOMT were stably expressed in the mammary epithelial cells of ovine.The effect of overexpressed melatonin on the anti-inflammatory and reproductive efficiency of ovine was studied as well.The transgenic Dorper and white Suffolk sheeps were produced through CRISPR/Cas9 combined with pronucleus microinjection technology.The linearized fragments of specific expression vector pBC1-AANAT and pBC1-HIOMT together with the Cas9 mRNA-sgRNA mixture were injected into pronucleus stage embryos.The integration with embryo genome was realized.Therefore,the transgenic ovine group carrying overexpressed melatonin synthetase gene AANAT and HIOMT was established.The sheep donors for superovulation come from 166 Dorper sheep and 139 white Suffolk sheep.In the superovulation,endoscopy was performed at 50-55h after the removal of vaginal plug,and the embryo was blanched after 10-12h.The total number of available embryos for Dorper sheep and white Suffolk sheep was 1989 and 1455,respectively,while the average available embryos for Dorper sheep and white Suffolk sheep were 11.98 and 10.47,respectively.After pronucleus embryos microinjection,1728 of total survival embryos for Dorper and 1321 for Suffolk were obtained.A total of 433 and 309 transplant recipients were used for Dorper and white Suffolk respectively.The overall pregnancy rate for Dorper was 39.03%(169/433),while it was 34.30%(106/309)for the white Suffolk.The positive individuals were identified by PCR-sequencing and Southern blot.It was proved that there were 87 sheep with AANAT and HIOMT transgenic positive.Among of them,there were 6 Dorper and 7 white Suffolk positive for mammary gland specific expression of AANAT,while there were 3 Dorper and 6 white Suffolk positive for mammary gland specific expression of HIOMT.The positive individuals for the co-expression of AANAT and HIOMT were 28 Dorper and 13 whited Suffolk.In the nonspecific expression sheep,14 AANAT and 10 HIOMT positive white Suffolk were detected.The positive rate was 15.54%(37/238)in Dorper sheep and 45.05%(50/111)in white Suffolk,while the overall positive rate was 24.93%(87/349).The systematic studies on the expression of melatonin synthase sheep in the breast of offspring were carried out.It was indicated that;1)the high level expression of exogenous genes AANAT and HIOMT in the mammary epithelial cells was achieved through inducing by prolactin in the lactation;2)the level of melatonin in the positive individual milk was significantly higher than that in the wild type,while there was no difference in the blood serum.It was illustrated that the enzymatic activity of the exogenous melatonin synthase genes,especially synthetase AANAT might be regulated by its circadian rhythm of the ovine;3)all kinds of physiological and biochemical indexes of blood and milk were not affected by inducing the melatonin synthetase gene AANAT and HIOMT.All of the indicators for the white blood cells,red blood cells and platelets were in the normal range and there were no difference from wild sheep;4)the efficiency of the superovulation for the transgenic sheep were higher than that for the wild type.It meant that more available embryos could be provided.The relative higher pregnancy rate and lambing rate could be achieved by the endoscope insemination in the propagation process;5)in the lipopolysaccharide(LPS)simulation experiment of mastitis induced by attack of bacteria,it was found that the attack bacteria LPS could quickly emerge the symptoms of mastitis.However,the transgenic sheep with the melatonin synthetase of the mammary gland specific expression showed a higher mastitis resistance than the wild type.The high level of melatonin in the mammary gland promoted the reduction of Il-2 and Il-8 levels in the white blood cells,and improved the autophagy level of monocytes to reduce the inflammatory response.It was concluded that the cloned melatonin AANAT and HIOMT synthetase genes which was promoted by the specific expressed casein promoter in the breast and the general expression of the cytomegalovirus(CMV)promoters respectively were expressed at a high level in vivo of the sheep.The breast overexpressed melatonin synthase,depending on the vector without marker genes and the prokaryotic fragments,could make the transgenic sheep safely produce the function milk of high content melatonin.The melatonin with high expression could not only improve the efficiency of the embryo transfer and the reproduction of the sheep efficiency,but also had a positive impact on the immune regulation and anti-inflammatory of the sheep themselves.The resuls of this study will establish the theoretical foundation of sheep breeding with high reproductivity and high disease resistance of sheep.